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Small hairpin RNA

About: Small hairpin RNA is a research topic. Over the lifetime, 9279 publications have been published within this topic receiving 285471 citations.


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Journal ArticleDOI
TL;DR: Results of the experiments indicated that hairpin dsRNA was just as effective as ds RNA in promoting the destruction of targeted mRNA, the EGFP marker could be expressed from the construct, and the distance of the SV40 intron from the inverted repeat was critical for the transcribed RNA to function in RNAi.

146 citations

Journal ArticleDOI
TL;DR: An efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids is demonstrated and a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms.
Abstract: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.

146 citations

Journal ArticleDOI
06 Mar 2008-Oncogene
TL;DR: The mechanism by which reduced Pdcd4 expression promotes invasion appears to involve the activation of β-catenin/Tcf and AP-1-dependent transcription.
Abstract: Programmed cell death 4 (Pdcd4) is a tumor suppressor that inhibits neoplastic transformation and tumor invasion. Tissue microarray analysis showed that Pdcd4 expression is downregulated in colon adenocarcinoma and carcinoma relative to adjacent normal tissues. To address the issue of whether reduced Pdcd4 expression is sufficient to promote tumor progression, we knocked down Pdcd4 expression in colon tumor HT29 cells using pdcd4 short hairpin RNA (shRNA). Pdcd4 knockdown results in a fibroblast-like transition, while the control cells (expressing LacZ shRNA) remain as clumped similar to the parental cells. In addition, expression of pdcd4 shRNA in HT29 cells promotes invasion. In an effort to characterize the molecular mechanism underlying these observations, we discovered that knockdown of Pdcd4 results in reduction of E-cadherin expression, and accumulation of active β-catenin in the nucleus. The active β-catenin binds with T-cell factor 4 (Tcf4) and activates β-catenin/Tcf-dependent transcription. Furthermore, Pdcd4 knockdown dramatically increases AP-1-dependent transcription. Thus, the mechanism by which reduced Pdcd4 expression promotes invasion appears to involve the activation of β-catenin/Tcf and AP-1-dependent transcription.

146 citations

Journal ArticleDOI
TL;DR: It is demonstrated that the impaired growth phenotype associated with the UL82 (pp71) deletion mutant is abolished when hDaxx knockdown cells are infected, suggesting that pp71 functions to relieve hDAXx-mediated repression during HCMV infection.
Abstract: This study examines the role of the cellular protein hDaxx in controlling human cytomegalovirus (HCMV) immediate-early (IE) gene expression and viral replication. Using permissive cell lines that either overexpress hDaxx or are depleted of hDaxx expression by the use of short hairpin RNA, we demonstrate that hDaxx functions as a repressor of HCMV IE gene expression and replication. In addition, we demonstrate that the impaired growth phenotype associated with the UL82 (pp71) deletion mutant is abolished when hDaxx knockdown cells are infected, suggesting that pp71 functions to relieve hDaxx-mediated repression during HCMV infection.

146 citations

Journal ArticleDOI
TL;DR: Findings indicate that TNFα and IL-1β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBPβ signaling.
Abstract: Objective. To investigate tumor necrosis factor α (TNF α) and interleukin-1β (IL-1 β) regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. Methods. Quantitative reverse transcription polymerase chain reaction and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB,CCAAT/enhancer binding protein (C/EBPβ), and MAPK on cytokine-mediated CCL3 promoter activity. The effect of NP-conditioned medium on macrophage migration was measured using a Transwell system. Results. An increase in CCL3 expression and promoter activity was observed in NP cells after TNF α or IL-1_ treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBP β on the CCL3 promoter was confirmed through gain-of-function and loss-of-function studies. Notably, cotransfection with p50 completely blocked cytokine and p65-dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with short hairpin RNA for p65 (shp65) and shIKKβ significantly decreased the TNF α dependent increase in CCL3 expression. Analysis of degenerated human NP tissue samples showed that CCL3, but not CCL4, expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNF α or IL-1 β promoted their migration. Pretreatment of macrophages with an antagonist of CCR1, the primary receptor for CCL3 and CCL4, blocked cytokine-mediated migration. Conclusion. Our findings indicate that TNF α and IL-1 β modulate the expression of CCL3 in NP cells by controlling the activation of MAPK, NF-κB, and C/EBP β signaling. The CCL3–CCR1 axis may play an important role in promoting macrophage infiltration in degenerated, herniated discs.

145 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023804
2022477
2021384
2020454
2019541
2018518